Angiotensin II alone or in combination for the treatment of hypotension

ABSTRACT

The present invention relates, inter alia, to a method comprising administering to a subject having high output shock and undergoing treatment with a catecholamine at a dose equivalent to at least about 0.2 mcg/kg/min of norepinephrine a dose of angiotensin II which is effective to raise the blood pressure of the subject to a mean arterial pressure (MAP) of about 65 mm Hg or above, and which is effective to reduce the dose of the catecholamine required to maintain a MAP of about 65 mm Hg to the equivalent of about 0.05-0.2 mcg/kg/min norepinephrine or less, or to the equivalent of about 0.05 mcg/kg/min norepinephrine or less.

This application is a continuation of U.S. patent application Ser. No.14/941,301, filed Nov. 13, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/575,127, filed on Dec. 18, 2014, and claims thebenefit of the filing date of U.S. Provisional Applications 61/917,576,filed Dec. 18, 2013, and U.S. Provisional Application 61/955,706, filedMar. 19, 2014, each of which is incorporated by reference in theirentirety herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2014, isnamed 123585_375374_SL.txt and is 2,088 bytes in size.

BACKGROUND INFORMATION

Critically ill patients with shock requiring vasopressors are at a highrisk of death. High output shock (also known as distributive shock) isthe most common form of shock, and is often caused by sepsis [1]. Whenshock is treated with vasopressors, two main classes of vasopressors arein the intensivists' armamentarium: catecholamines and vasopressin typepeptides [1]. Currently, no specific type of vasopressor (e.g.norepinephrine, vasopressin, dopamine) compared to another vasopressorhas been shown to improve outcome [2]. All vasopressors have limitationsand potential side effects. Patients treated with catecholamines forshock often develop tachyphylaxis thereby limiting the utility of theseagents, and high doses of catecholamines can cause direct cardiotoxicity[3]. The toxic potential of catecholamines has been recentlydemonstrated in a randomized clinical trial of septic shock patientstreated norepinephrine [4]. In this study, beta-blockade with esmololwas shown to improve survival in these patients by decreasing the heartrate. Thus, vasopressors that are not inotropes or chronotropes may beuseful in patients with shock. One such vasopressor is vasopressin,which is most commonly used as an adjuvant with catecholamines.Vasopressin has been shown to improve outcomes in patients with lesssevere septic shock, but has toxicity (e.g. cardiac and mesentericischemia) at high doses and interacts with hydrocortisone [5]. Inhigh-output shock, the patients are critically ill and mean arterialpressure cannot be maintained without vasopressors. High-output shock isdefined as a cardiovascular Sequential Organ Function Assessment (SOFA)score of greater than or equal to 3 or 4 as well as a cardiac indexof >2.4 liters/min/BSA 1.73 m² [10]. In high-output shock, if bloodpressure cannot be maintained, it is uniformly fatal. In patients thatcannot maintain their blood pressure, the addition of a ‘rescue’vasopressor in this setting could be useful.

A subset of patients with shock (including high-output shock and othertypes of shock) are catecholamine-resistant. That is, they areunresponsive (do not exhibit an appropriate increase in blood pressure)in response to treatment with a dose of a catecholamine equivalent to adose of at least 0.2 mcg/kg/min of norepinephrine.

Angiotensin II (sometimes referred to herein as ATII) is a naturallyoccurring peptide hormone with endocrine, autocrine, paracrine, andintracrine hormonal effects. It is a potent direct vasoconstrictor,constricting both arteries and veins and increasing blood pressure [6].It has a half-life in circulation of approximately 30 seconds, but whilein tissue, its half-life may be as long as 15-30 minutes. ATII increasessecretion of ADH and ACTH, and may potentiate sympathetic effects bydirect action on postganglionic sympathetic fibers. It also acts on theadrenal cortex, causing it to release aldosterone [6,7]. High doses ofangiotensin II have been reported to induce adverse side effects,including for example, mesenteric ischemia and bronchospasm.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows study drug titration protocol.

FIG. 2 shows patient flow diagram.

FIG. 3 shows changes in norepinephrine dose with concurrent angiotensinII.

FIG. 4 shows Angiotensin II Dose Titration—Quintiles

DESCRIPTION

This invention relates, inter alia, to the surprising finding by thepresent inventors that in response to the administration of very lowdoses of angiotensin II to subjects having hypotension, e.g., exhibitingdistributive shock (high output shock), the blood pressure can be raisedto a normal level (e.g., a mean arterial pressure (MAP) of about 65 mmHg or higher) and can be maintained at this level, even in the absenceof, or with low doses of, other agents such as vasopressin orcatecholamines (e.g., norepinephrine) that are generally administered tosuch subjects as the standard of care. The reduction or elimination of aneed to administer a catecholamine (e.g., norepinephrine) is sometimesreferred to herein as a catecholamine-sparing (norepinephrine-sparing)effect. Administering low doses of angiotensin II and of a catecholaminesuch as norepinephrine reduces undesirable side effects brought about bythese drugs. High doses of catecholamines can be toxic, and the bluntingof these toxic effects has been associated with improved survival inpatient with shock. Maintenance of blood pressure during shock iscritical to survival. In addition to raising blood pressure, heart rateand hemodynamics are improved or remain stable following administrationof the low doses of angiotensin II.

Definitions

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,treatment with “a” catecholamine as used above includes treatment withone or more types of catecholamine.

The term “about” as used herein, means within about 10% of the indicatedvalue, preferably plus or minus 5% of the indicated value.

The term “angiotensin II” may refer to Asp-Arg-Val-Tyr-Ile-His-Pro-Phe[SEQ ID NO: 1] also called 5-isoleucine angiotensin II. SEQ ID NO: 1 isan octa-peptide naturally present in humans and other species, such asequines, hogs, etc. Isoleucine may be substituted by valine to result in5-valine angiotensin II, Asp-Arg-Val-Tyr-Val-His-Pro-Phe [SEQ ID NO: 2].Other angiotensin II analogues such as [Asn¹-Phe⁴]-angiotensin II [SEQID NO: 3], hexapeptide Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 4],nonapeptide Asn-Arg-Val-Tyr-Tyr-Val-His-Pro-Phe [SEQ ID NO: 5],[Asn¹-Ileu⁵-Ileu⁸]-angiotensin II [SEQ ID NO: 6],[Asn¹-Ileu⁵-Ala⁸]-angiotensin II [SEQ ID NO: 7], and[Asn¹-diiodoTyr⁴-Ileu⁵]-angiotensin II [SEQ ID NO: 8] may also be used.Angiotensin II may be synthesized, for example, by solid phase peptidesynthesis to incorporate modifications, such as C-terminal amidation.C-terminal acetate groups may also be added. The term “angiotensin II”,without further specificity, is intended to refer to any of thesevarious forms, as well as combinations thereof.

The term “catecholamine”, as used herein, refers to dopamine,norepinephrine, epinephrine, phenylephrine, ephedrine and theirprodrugs, structural analogs, or derivatives that induce similarphysiological effects in humans, e.g., raise mean arterial pressure inhealthy human subjects. In certain embodiments, the catecholamine may bedopamine, norepinephrine, epinephrine, ephedrine or phenylephrine.

The term “catecholamine-resistant hypotension” as used herein refers topatients who require more than 15 μg/kg/min of dopamine, 0.1 μg/kg/minnorepinephrine, or 0.1 μg/kg/min epinephrine as a vasopressor. Dopamine,norepinephrine, and epinephrine may be administered at rates higher than15 μg/kg/min, 0.1 μg/kg/min, or 0.1 μg/kg/min, respectively, butelevated rates correlate with increased mortality.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The term “mean arterial pressure” or “MAP” refers to the averagearterial pressure during a single cardiac cycle.

As used herein, a “subject” or “patient” refers to any animal (e.g., amammal), including human, non-human primate, rodent, etc., which is tothe recipient of a particular treatment. Typically, the terms “subject”and “patient” are used interchangeable herein in reference to a humansubject.

Angiotensin II Therapeutics

Angiotensin II is a peptide hormone naturally produced by the body thatregulates blood pressure via vasoconstriction and sodium reabsorption.Hemodynamic effects of angiotensin II administration have been thesubject of numerous clinical studies, demonstrating significant effectson systemic and renal blood flow (Harrison-Bernard, L. M., The renalrenin-angiotensin system. Adv Physiol Educ, (2009) 33(4): p. 270-74).Angiotensin II is a hormone produced by the renin angiotensinaldosterone system (RAAS) that modulates blood pressure via regulationof vascular smooth muscle tone and extracellular fluid homeostasis.Angiotensin II mediates its effects on the vasculature by inducingvasoconstriction and sodium retention. In addition to its systemiceffects, angiotensin II has a pronounced effect on the efferentarterioles of the kidney, maintaining glomerular filtration when bloodflow is decreased. Angiotensin II also regulates sodium reabsorption inthe kidney by stimulating Na+/H+ exchangers in the proximal tubule andinducing the release of aldosterone and vasopressin (Harrison-Bernard,L. M., The renal renin-angiotensin system. Adv Physiol Educ, 2009.33(4): p. 270-4.).

The sequence of angiotensin II used in the compositions and methodsdisclosed herein may be homologous to the sequences of angiotensin IIdescribed above. In certain aspects, the invention includes isolated,synthetic, or recombinant amino acid sequences that are at least 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, 2, 3, 4,5, 6, 7, and/or 8. Any such variant sequences may be used in place of anangiotensin II as described in the preceding paragraph.

In some aspects, the angiotensin II may be selected from 5-valineangiotensin II, 5-valine angiotensin II amide, 5-L-isoleucineangiotensin II, and 5-L-isoleucine angiotensin II amide, or apharmaceutically acceptable salt thereof, preferably manufactured undercurrent good manufacturing conditions (cGMP). In some aspects, thecomposition may include different forms of angiotensin II in differentpercentages, e.g., a mixture of hexapeptide and nonapeptide angiotensin.The composition comprising angiotensin II may be suitable for parenteraladministration, e.g., for injection or intravenous infusion.

Similarly, an angiotensin II therapeutic may be used as any suitablesalt, deprotected form, acetylated form, deacetylated form, and/orprodrug form of the above-mentioned peptides, including pegylated formsof the peptides or conjugates as disclosed in U.S. Pat. No. 7,666,408(incorporated by reference). The term “prodrug” refers to any precursorcompound which is able to generate or to release the above-mentionedpeptide under physiological conditions. Such prodrugs may be largerpeptides which are selectively cleaved in order to form the peptide ofthe invention. For example, in some aspects, the prodrug may beangiotensin I or its homologues that may result in angiotensin II by theaction of certain endogenous or exogenous enzymes. Further prodrugsinclude peptides with protected amino acids, e.g., having protectinggroups at one or more carboxylic acid and/or amino groups. Suitableprotecting groups for amino groups are the benzyloxycarbonyl,t-butyloxycarbonyl (BOC), fluorenylmethyloxycarbonyl (FMOC), formyl, andacetyl or acyl group. Suitable protecting groups for the carboxylic acidgroup are esters such as benzyl esters or t-butyl esters. The presentinvention also contemplates the use of angiotensin II and/or precursorpeptides having amino acid substitutions, deletions, additions, thesubstitutions and additions including the standard D and L amino acidsand modified amino acids, such as, for example, amidated and acetylatedamino acids, wherein the therapeutic activity of the base peptidesequence is maintained at a pharmacologically useful level.

Indications

Methods of the invention can be used to treat a subject exhibiting avariety of types of shock, such as, e.g., high output shock, septicshock or shock from cardiac arrest or cardiogenic shock. Otherconditions that can be treated with the indicated low doses ofangiotensin II include acute kidney injury (AKI), hepato-renal syndrome(HRS) and variceal bleeding.

Doses of the Therapeutically Effective Substance

In general, angiotensin II increases blood pressure, and patients whoare hypotensive may require larger doses to exhibit pressor responsessimilar to those observed in normal patients. The composition includingthe angiotensin therapeutic (e.g., angiotensin II) can be administeredat a rate sufficient to achieve a target blood pressure. For example, apatient may be coupled to a monitor that provides continuous, periodic,or occasional measurements of MAP.

The precise amount of a drug to be administered to a mammal for thetreatment of hypotension and shock is dependent on numerous factorsknown to one skilled in the art, such as, the agent to be administered,the general condition of the patient, the condition to be treated, thedesired duration of use, the type of mammal, the method ofadministration etc.

The dose of angiotensin II can be administered at a rate of from about0.25 ng/kg/min to about 100 ng/kg/min, e.g. from about 10 ng/kg/min toabout 50 ng/kg/min, from about 20 ng/kg/min to about 40 ng/kg/min, fromabout 0.25 ng/kg/min to about 20 ng/kg/min, from about 0.25 ng/kg/min toabout 10 ng/kg/min, from about 0.25 ng/kg/min to about 5 ng/kg/min, fromabout 1.25 ng/kg/min to about 20 ng/kg/min, about 1.25 ng/kg/min toabout 10 ng/kg/min, or from about 1.25 ng/kg/min to about 5 ng/kg/min.In embodiments of the invention, the dose is about 0.25 ng/kg/min, about0.5 ng/kg/min, about 1 ng/kg/min, about 1.25 ng/kg/min, about 1.5ng/kg/min, about 2 ng/kg/min, about 2.5 ng/kg/min, about 3 ng/kg/min,about 3.5 ng/kg/min, about 4 ng/kg/min, about 4.5 ng/kg/min, about 5ng/kg/min, about 5.5 ng/kg/min, about 6 ng/kg/min, about 7.5 ng/kg/minor about 10 ng/kg/min.

FIG. 4 and the Examples indicate that subjects with high output shockrequiring high doses of catecholamine (in the exemplified case,norepinephrine) who are administered as little as 1 or 2.5 ng/kg/min ofangiotensin II show an increase in blood pressure and maintain it, evenin the absence of, or with very low doses of, norepinephrine. In thestudy illustrated in FIG. 4, subjects requiring high doses ofnorepinephrine (>0.2 mcg/kg/min) responded to low dose angiotensin II,such that the norepinephrine was discontinued, and the effect ofangiotensin II converted these patients from hypotensive tohypertensive.

The dose administration can last from about 0.25 hours to about 120hours, e.g., from about 1 hour to about 7 hours, 2 hours to about 6hours, or about 3 hours to about 5 hours.

The therapeutic regimen can be started within, e.g., 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, or 72 hours, after theonset of acute symptoms.

Formulations

Suitable formulations (pharmaceutical compositions) for administering adrug will depend on the mode of administration. For example,formulations adapted for parenteral administration may comprise asterile aqueous preparation, preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using suitable dispersing or wetting agents and suspendingagents. Illustrative of a preparation produced in such conventionalfashion is the aqueous formulation, Remestyp (terlipressin). Thepreparation also may be a sterile injectable solution or suspension in adiluent or solvent, for example as a solution in 1,3-butanediol, water,Ringer's solution, and isotonic sodium chloride solution, which areexemplary acceptable diluents. Sterile, fixed oils may be employed as asolvent or suspending medium. Bland fixed oils, including synthetic monoor di-glycerides, and fatty acids, such as oleic acid, may also be used.Most of the agents described herein are commercially available and canbe obtained readily from commercial sources.

Excipients

The pharmaceutical compositions of the present invention may alsocontain diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable carrier” refers to a non-toxic carrier that may beadministered to a patient, together with a therapeutically effectivesubstance (such as angiotensin II) of this invention, and which does notdestroy the pharmacological activity of the therapeutically effectivesubstance. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. The term“excipient” refers to an additive in a formulation or composition thatis not a pharmaceutically active ingredient.

One of skill in the art would appreciate that the choice of any oneexcipient may influence the choice of any other excipient. For example,the choice of a particular excipient may preclude the use of one or moreadditional excipients because the combination of excipients wouldproduce undesirable effects. One of skill in the art would be able toempirically determine which excipients, if any, to include in thecompositions of the invention. Excipients of the invention may include,but are not limited to, co-solvents, solubilizing agents, buffers, pHadjusting agents, bulking agents, surfactants, encapsulating agents,tonicity-adjusting agents, stabilizing agents, protectants, andviscosity modifiers. In some aspects, it may be beneficial to include apharmaceutically acceptable carrier in the compositions of theinvention.

Solubilizing Agents

In some aspects, it may be beneficial to include a solubilizing agent inthe compositions of the invention. Solubilizing agents may be useful forincreasing the solubility of any of the components of the formulation orcomposition, including a therapeutically effective substance (e.g.,angiotensin II) or an excipient. The solubilizing agents describedherein are not intended to constitute an exhaustive list, but areprovided merely as exemplary solubilizing agents that may be used in thecompositions of the invention. In certain aspects, solubilizing agentsinclude, but are not limited to, ethyl alcohol, tert-butyl alcohol,polyethylene glycol, glycerol, methylparaben, propylparaben,polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceuticallyacceptable salts and/or combinations thereof.

pH-Adjusting Agents

In some aspects, it may be beneficial to adjust the pH of thecompositions by including a pH-adjusting agent in the compositions ofthe invention. Modifying the pH of a formulation or composition may havebeneficial effects on, for example, the stability or solubility of atherapeutically effective substance, or may be useful in making aformulation or composition suitable for parenteral administration.pH-adjusting agents are well known in the art. Accordingly, thepH-adjusting agents described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary pH-adjustingagents that may be used in the compositions of the invention.pH-adjusting agents may include, for example, acids and bases. In someaspects, a pH-adjusting agent includes, but is not limited to, aceticacid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodiumcarbonate, and combinations thereof.

The pH of the compositions of the invention may be any pH that providesdesirable properties for the formulation or composition. Desirableproperties may include, for example, therapeutically effective substance(e.g., angiotensin II) stability, increased therapeutically effectivesubstance retention as compared to compositions at other pHs, andimproved filtration efficiency. In some aspects, the pH of thecompositions of the invention may be from about 3.0 to about 9.0, e.g.,from about 5.0 to about 7.0. In particular aspects, the pH of thecompositions of the invention may be 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1,5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1, or 6.5±0.1.

Buffers

In some aspects, it may be beneficial to buffer the pH by including oneor more buffers in the compositions. In certain aspects, a buffer mayhave a pKa of, for example, about 5.5, about 6.0, or about 6.5. One ofskill in the art would appreciate that an appropriate buffer may bechosen for inclusion in compositions of the invention based on its pKaand other properties. Buffers are well known in the art. Accordingly,the buffers described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary buffers that maybe used in the compositions of the invention. In certain aspects, abuffer may include one or more of the following: Tris, Tris HCl,potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate,combinations of sodium and potassium phosphate, Tris/Tris HCl, sodiumbicarbonate, arginine phosphate, arginine hydrochloride, histidinehydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonicacid (MES), maleate, bis-tris, phosphate, carbonate, and anypharmaceutically acceptable salts and/or combinations thereof.

Surfactants

In some aspects, it may be beneficial to include a surfactant in thecompositions of the invention. Surfactants, in general, decrease thesurface tension of a liquid composition. This may provide beneficialproperties such as improved ease of filtration. Surfactants also may actas emulsifying agents and/or solubilizing agents. Surfactants are wellknown in the art. Accordingly, the surfactants described herein are notintended to constitute an exhaustive list, but are provided merely asexemplary surfactants that may be used in the compositions of theinvention. Surfactants that may be included include, but are not limitedto, sorbitan esters such as polysorbates (e.g., polysorbate 20 andpolysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides andpolyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g.,lecithin), and combinations thereof.

Tonicity-Adjusting Agents

In some aspects, it may be beneficial to include a tonicity-adjustingagent in the compositions of the invention. The tonicity of a liquidcomposition is an important consideration when administering thecomposition to a patient, for example, by parenteral administration.Tonicity-adjusting agents, thus, may be used to help make a formulationor composition suitable for administration. Tonicity-adjusting agentsare well known in the art. Accordingly, the tonicity-adjusting agentsdescribed herein are not intended to constitute an exhaustive list, butare provided merely as exemplary tonicity-adjusting agents that may beused in the compositions of the invention. Tonicity-adjusting agents maybe ionic or non-ionic and include, but are not limited to, inorganicsalts, amino acids, carbohydrates, sugars, sugar alcohols, andcarbohydrates. Exemplary inorganic salts may include sodium chloride,potassium chloride, sodium sulfate, and potassium sulfate. An exemplaryamino acid is glycine. Exemplary sugars may include sugar alcohols suchas glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

Stabilizing Agents

In some aspects, it may be beneficial to include a stabilizing agent inthe compositions of the invention. Stabilizing agents help increase thestability of a therapeutically effective substance in compositions ofthe invention. This may occur by, for example, reducing degradation orpreventing aggregation of a therapeutically effective substance. Withoutwishing to be bound by theory, mechanisms for enhancing stability mayinclude sequestration of the therapeutically effective substance from asolvent or inhibiting free radical oxidation of the anthracyclinecompound. Stabilizing agents are well known in the art. Accordingly, thestabilizing agents described herein are not intended to constitute anexhaustive list, but are provided merely as exemplary stabilizing agentsthat may be used in the compositions of the invention. Stabilizingagents may include, but are not limited to, emulsifiers and surfactants.

Modes of Administration

Administration of angiotensin II or a catecholamine can be by anyconvenient route, e.g. intravenous (using either a bolus or by a steadyinfusion), intramuscular, subcutaneous or inhalation. The angiotensin IIand the catecholamine may be administered together or independently.

The compositions of the invention can be administered in a variety ofconventional ways. In some aspects, the compositions of the inventionare suitable for parenteral administration. These compositions may beadministered, for example, intraperitoneally, intravenously,intrarenally, or intrathecally. In some aspects, the compositions of theinvention are injected intravenously. One of skill in the art wouldappreciate that a method of administering a therapeutically effectivesubstance formulation or composition of the invention would depend onfactors such as the age, weight, and physical condition of the patientbeing treated, and the disease or condition being treated. The skilledworker would, thus, be able to select a method of administration optimalfor a patient on a case-by-case basis.

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature andtechniques relating to chemistry, molecular biology, cell and cancerbiology, immunology, microbiology, pharmacology, and protein and nucleicacid chemistry, described herein, are those well-known and commonly usedin the art.

Methods Related to the Administration of Angiotensin II

One aspect of the invention is a method comprising administering to asubject having high output shock (e.g., catecholamine-resistant highoutput shock) and undergoing treatment with a catecholamine at a doseequivalent to at least about 0.2 mcg/kg/min of norepinephrine a dose ofangiotensin II which is effective to raise the blood pressure of thesubject to a mean arterial pressure (MAP) of about 65 mm Hg or above,and which is effective to reduce the dose of the catecholamine requiredto maintain a MAP of about 65 mm Hg to the equivalent of about 0.05-0.2mcg/kg/min norepinephrine or less.

Another aspect of the invention is a method comprising administering toa subject having high output shock (e.g., catecholamine-resistant highoutput shock) and undergoing treatment with a catecholamine at a doseequivalent to at least about 5 mcg/min of norepinephrine a dose ofangiotensin II which is effective to raise the blood pressure of thesubject to a mean arterial pressure (MAP) above about 80 mm Hg, andwhich is effective to reduce the dose of the catecholamine required tomaintain a MAP above about 80 mm Hg to the equivalent of less than about5-10 mcg/min norepinephrine. In certain such embodiments, the dose ofangiotensin II is at least about 20 ng/kg/min.

In various embodiments, the dose of angiotensin II is effective toreduce the dose of the catecholamine required to maintain a MAP of about65 mm Hg to the equivalent of about 0.05 mcg/kg/min norepinephrine orless.

In embodiments of the invention,

-   a. the catecholamine is norepinephrine, or-   b. the catecholamine is epinephrine and the dose equivalent to 0.1    mcg/kg/min of norepinephrine is 0.1 mcg/kg/min; or-   c. the catecholamine is dopamine and the dose equivalent to 0.1    mcg/kg/min of norepinephrine is 15 mcg/kg/min; or-   d. the catecholamine is phenylephrine and the dose equivalent to 0.1    mcg/kg/min of norepinephrine is 1.0 mcg/kg/min.

In embodiments of the invention, the dose of angiotensin II is fromabout 0.25 ng/kg/min to about 10 ng/kg/min, from about 0.25 ng/kg/min toabout 5 ng/kg/min; about 1 ng/kg/min; about 2 ng/kg/min or about 3ng/kg/min.

Another aspect of the invention is a method comprising administering toa subject having high output shock (e.g., catecholamine-resistant highoutput shock) and undergoing treatment with a catecholamine at a doseequivalent to at least about 0.2 mcg/kg/min of norepinephrine a dose ofangiotensin II which is effective to raise the blood pressure of thesubject to a MAP of about 65 mm Hg or above, and which is effective toreduce the dose of the catecholamine required to maintain a MAP of about0.05 mcg/kg/min norepinephrine or less, which further comprises

identifying the subject (selecting a subject) as belonging to the subsetof subjects who are sufficiently responsive to angiotensin II so that adose of angiotensin II of about 0.25 ng/kg/min to about 5 ng/kg/min iseffective to reduce the amount of norepinephrine required to maintain amean arterial pressure (MAP) at or above about 65 mm Hg to about 0.05mcg/kg/min epinephrine or less, by titrating the amount of angiotensinwith regard to a fixed dose of norepinephrine; and

continuing to administer angiotensin II to the subject, at the dosedetermined as above.

In various methods of the invention, the angiotensin II and thecatecholamine may be administered intravenously, intramuscularly,subcutaneously or by inhalation; and they may be administered togetheror independently.

Another aspect of the invention is a method comprising administering toa subject having shock (e.g., septic shock or shock from other causes,such as cardiac arrest or cardiogenic shock) and undergoing treatmentwith a catecholamine at a dose equivalent to at least about 0.2mcg/kg/min of norepinephrine a dose of angiotensin II which is effectiveto raise the blood pressure of the subject to a MAP of about 65 mm Hg orabove, and which is effective to reduce the dose of the catecholaminerequired to maintain a MAP of about 65 mm Hg to the equivalent of about0.05-0.2 mcg/kg/min norepinephrine or less, or to the equivalent ofabout 0.05 mcg/kg/min norepinephrine or less.

In some methods of the invention, the subject is a human.

In certain embodiments, the angiotensin II and the catecholamine areconjointly administered. As used herein, the phrase “conjointadministration” refers to any form of administration of two agents suchthat the second agent is administered while the previously administeredagent is still effective in the body (e.g., the two agents aresimultaneously effective in the patient, which may include synergisticeffects of the two agents). For example, the two agents can beadministered either in the same formulation or in separate formulations,either concomitantly or sequentially. In certain embodiments, thedifferent agents can be administered within one hour, 12 hours, 24hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, anindividual who receives such treatment can benefit from a combinedeffect of the two agents.

In various methods of the invention, the subject is undergoingstandard-of-care treatment with a catecholamine (e.g., epinephrine,norepinephrine, dopamine, phenylephrine, ephedrine) or with vasopressinat or prior to the time the angiotensin II is administered. These agentsare administered at a dose which is equivalent to at least 0.2mcg/kg/min of the catecholamine, norepinephrine. Typical equivalentdoses are:

Drug Dose Norepinephrine Equivalent Epinephrine 0.1 mcg/kg/min 0.1mcg/kg/min Norepinephrine 0.1 mcg/kg/min 0.1 mcg/kg/min Dopamine 15mcg/kg/min 0.1 mcg/kg/min Phenylephrine 1.0 mcg/kg/min 0.1 mcg/kg/minVasopressin 0.04 U/min 0.1 mcg/kg/min

In some embodiments, the patients are catecholamine-resistant. That is,the patients are not responsive (do not exhibit an increase in bloodpressure) to a catecholamine administered in a dose that is equivalentto a dose of at least about 0.2 mcg/kg/min of norepinephrine.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Theembodiments described herein are not intended as limitations on thescope of the invention. The present description is further illustratedby the following examples, which should not be construed as limiting inany way. In the foregoing and in the following examples, alltemperatures are set forth in uncorrected degrees Celsius; and, unlessotherwise indicated, all parts and percentages are by weight.

EXAMPLES Example I—Intravenous Angiotensin II for the Treatment of HighOutput Shock

A. Methods

Study Patients

Patients were older than 21 years of age and deemed to have high outputshock, defined as a cardiovascular Sequential Organ Function Assessment(SOFA) score of 4 as well as a cardiac index of >2.4 liters/min/BSA 1.73m² [10]. Patients also had an indwelling arterial line and urinarycatheter as part of standard care. In addition, each subject was foundto be adequately volume resuscitated and clinically assessed not to bevolume responsive (i.e. a fluid bolus would fail to increase cardiacindex by 15%). Standard of care was to resuscitate with 20-30 cc/kg ofcrystalloid as initial resuscitation. Exclusion criteria includedpatients with acute coronary syndrome, a known history of vasospasm orasthma, any patients currently experiencing bronchospasm or patientswith active bleeding with an anticipated need for transfusion of >4units of packed red blood cells, hemoglobin <7 g/dL or any othercondition that would contraindicate drawing serial blood samples.

Treatment Assignments

Upon enrollment in the study, patients were randomly assigned thefollowing randomization procedures (computerized random numbers) toreceive either angiotensin II acetate infusion (Clinalfa, Bachem A G,Hauptstrasse 144, 4416 Bubendorf, Switzerland) or a placebo infusion(hereafter referred to as the Study Drug and placebo, respectively). Theinvestigators, clinical support staff, the patients and their familieswere unaware of the treatment assignment for the duration of the study.

Drug Infusion

Enrolled patients were randomized to receive the Study Drug infusion innormal saline calculated to run at a drip rate corresponding to aninitial concentration of 20 ng/kg/min, plus the standard-of-caretreatment for high output shock. The Study Drug was prepared in anopaque cellophane bag, the contents of which were unknown to theinvestigators, nurses or anyone else taking direct care of the patient.The Study Drug was administered for a total of 6 hours, with dose (andcorresponding drip rate) adjustments made hourly. Study Drug doseadjustments were determined per a pre-specified protocol, based on theconcomitant requirements of standard-of-care therapy (in all cases,norepinephrine infusion plus vasopressin, epinephrine and/orphenylephrine infusions) needed to maintain a mean arterial pressure(MAP) at or above 65 mm Hg, which is the standard practice at ourinstitution. The Study Drug titration protocol was designed to elucidatethe dose of ATII that was required (in conjunction with a norepinephrinedose between 5-10 mcg/min) to achieve the aforementioned standard MAPgoal of 65 mm Hg. The dose titration protocol is shown on FIG. 1. Themaximum allowable dose for the ATII titration was 40 ng/kg/min, and theminimum was 5 ng/kg/min. At the end of 6 hours, the Study Drug infusionwas titrated off by being halved every 10 minutes until the Study Druginfusion dose was below 5 ng/kg/min, after which it was discontinued.

End Points

The primary endpoint was the effect of the ATII infusion on the standingdose of norepinephrine which was required to maintain a MAP of 65 mmHg.The secondary endpoints included the effect of the ATII infusion onurine output, serum lactate, cardiac output, and 30 day mortality.

Statistical Analysis

A small cohort of patients was analyzed, consistent with similar studiesof this nature. A population of 20 patients, ten patients in each arm,was determined to generate a basis for determining if ATII could affectthe dose of norepinephrine at the doses outlined herein. An independentdata and safety monitor (DSM) was assigned and reviewed all adverseevents.

The distribution of demographic and clinical variables was alsoassessed. Differences between proportions of patients with certainvariables were assessed with the chi-square, Fisher exact test, studentt, and Mann-Whitney test as appropriate. The primary endpoint of theeffect of the Study Drug infusion on the standing dose of norepinephrinewas calculated using a general estimating equation analysis and ispresented as the mean dose of norepinephrine (mcg/min) and Study Druginfusion (in ng/kg/min) at hourly intervals.

Generalized estimating equation was used to model the response to theStudy Drug over time, with standard-of-care vasopressor hourly readingsbeginning at 1 hour prior to, through 8 hours after the initiation ofthe Study Drug, using the SAS Genmod procedure (version 9.3, Cary,N.C.). Correlation structure was defined as auto-regressive to accountfor the likely higher correlation between time points that were closertogether. In this model, the main effect of drug examines the meanresponse to each drug averaged across times. The main effect of timeexamines the mean response at each time point averaged across drugs, andthe drug multiplied by time interaction examines whether the change overtime differs between drugs.

All values are reported as mean±standard deviation unless otherwisespecified. All other statistical analysis was completed using SPSS 18,Chicago, Ill., USA.

B. Results

The flow of patients into the study is reported in FIG. 2. Twentypatients underwent randomization and all 20 patients were enrolled inand completed the study (FIG. 1). Baseline characteristics of the twogroups are shown in Table 1. The mean age for all study subjects was62.9±15.8 years. Of the patients, 75% were male, 45% were Caucasian and40% were African American. Baseline SOFA and APACHE II scores were15.9±3.0 and 30.6±8.9, respectively. 19 of 20 patients were receivingconcomitant vasopressin at a dose of 0.02-0.08 u/min. Vasopressin doseswere not adjusted during the study period

ATII resulted in a reduction in norepinephrine dosing in all patients(FIG. 3). The mean Hour 1 norepinephrine dose for the placebo cohort was27.6±29.3 mcg/min v. 7.4±12.4 mcg/min for the ATII cohort (p=0.06). Hour2 norepinephrine dosing for the placebo cohort was 28.6±30.2 mcg/min v.7.3±11.9 mcg/min in the ATII cohort (p=0.06). Throughout the studyperiod, the mean ATII dose was reduced from 20 ng/kg/min at Hour Zero to5 ng/kg/min at Hour 6 before being titrated off by Hour 7 (one hourpost-infusion). Despite this down-titration of ATII, norepinephrinedoses remained substantially lower in the ATII cohort than the placebocohort, though the effect approached statistical significance only atHours 1 and 2. Upon cessation of the ATII infusion, mean norepinephrinerebounded concomitantly.

Using a general estimating equation model with time defined as acontinuous variable, in order to obtain a global test of interactioneffect, the main effect of treatment (Study Drug vs. placebo) was notsignificant (p=0.13), nor was the effect of time (p=0.30), nor was thetreatment multiplied by time interaction (p=0.76). When time was definedas a class variable with Hour −1 defined as the reference group, inorder to examine specific time points, the drug effect (p=0.14) and timeeffect (p=0.18 at time 0, p=0.51 at time 1) both remainednon-significant. The product of drug multiplied by time interactionshowed a trend level of significance at 1-hr and 2-hr (p=0.06).

Adverse events most commonly experienced by all patients were metabolicdisorders with alkalosis occurring in four patients in the ATII groupand zero in the placebo group (p=0.09). The most common adverse eventthought to be attributable to ATII was hypertension,

which occurred in 20% of patients receiving ATII (p=0.58). In both ofthese patients, the Study Drug infusion was stopped, per protocol, inorder to achieve MAP goals. Table 2 lists adverse events

Urine output, cardiac output, central venous pressure, and mean arterialpressure are shown in Table 3. The 30 day mortality for the two groupswere similar for the ATII cohort and the placebo cohort (50% v. 60%,p=1.00).

TABLE 1 Baseline demographic and clinical data Full P Cohort SD ATII SDPlacebo SD value 

Age 62.85 15.81 68.40 17.46 57.30 12.44 0.12 Male (n) 15 6 9 0.30 Race(n) Caucasian 9 6 3 0.37 Black 8 3 5 0.65 Other 3 1 2 1.00 Severity ofIllness Baseline SOFA 15.90 2.97 14.9 2.81 16.90 2.92 0.14 APACHE 30.608.86 27.2 9.67 34.00 6.83 0.09 Past Medical History IHD 2 1 1 1.00 CHF 22 0 0.47 COPD 2 2 0 0.47 DM 7 4 3 1.00 CKD 7 3 4 1.00 HD 1 0 1 1.00Liver disease 9 5 4 1.00 Cancer 6 1 5 0.14 IS 6 1 5 0.14 Steroids 3 1 21.00 Hypertension 9 4 5 1.00 CVA 5 4 1 0.30 AKI 17 9 8 1.00 Labs WBC17.38 19.0 16.0 15.72 12.3 0.61 Hgb 9.45 9.16 2.14 9.73 2.45 0.59Creatinine 2.33 1.89 1.03 2.76 1.34 0.12 pH 7.33 7.34 0.11 7.32 0.120.63 Lactate 5.83 4.59 3.11 7.06 5.16 0.21 Baseline vasopressordoses^(Y) Norepinephrine 25.05 17.03 19.80 11.67 30.30 20.37 0.18Vasopressin 0.04 0.02 0.03 0.02 0.05 0.02 0.10Results are presented as mean and SD or number. ¹P values for continuousvariables calculated using Students T test. P values for discretevariables calculated using Fisher exact test. ²One patient in theplacebo group received phenylephrine infusion prior to initiation ofATII versus no patients in the ATII group. One patient in the placebogroup received epinephrine versus no patients in the ATII group. SOFA,sequential organ function assessment; APACHE, acute physiology anchronic health evaluation II; IHD=ischemic heart disease. CHF=congestiveheart failure. COPD=chronic obstructive pulmonary disease. DM=diabetesmellitus. CKD=chronic kidney disease. HD=hemodialysis.IS=immunocompromised state. CVA=cerebrovascular accident. AKI=acutekidney injury; Hgb, hemoglobin; NA, to represent not analyzed, notapplicable, or not available.

TABLE 2 Adverse events Organ System Total ATII Placebo P value Metabolicdisorders 16 11 5 Acidosis 2 3 1.00 Alkalosis 4 0 0.09 Blood orlymphatic disorders 7 3 4 Respiratory disorders 6 3 3 Worseningrespiratory failure 1 3 0.58 Wheezing 1 0 1.00 Cardiac disorders 12 7 5Hypertension^(⋄) 2 0 0.58 Hypotension 2 1 1.00 Atrial Fibrillation 2 00.47 Renal disorders^(Y) 7 6 1 Decreased urine output 3 1 0.58 WorseningAKI 0 2 0.47 Other disorders 

8 5 3 Worsening MOSF 2 3 1.00 ^(⋄)ATII infusion was discontinued in twopatients due to hypertension. ^(Y)Seventeen of 20 patients exhibitedpre-existing AKI, including 8 patients receiving placebo and 9 patientsreceiving ATII. Of the three patients that did not have pre-existingAKI, one patient developed AKI and received ATII.

 Includes worsening multiple organ system failure, fever, lowerextremity edema, and thigh hematoma. AKI = acute kidney injury. MOSF =multiple organ system failure. P values calculated using Fisher Exacttest.

Urine output, cardiac output, central venous pressure, and mean arterialpressure are shown in Table 3. The 30 day mortality for the two groupswere similar for the ATII cohort and the placebo cohort (50% v. 60%,p=1.00).

TABLE 3 Secondary outcomes Hr-2 Hr-1 Hr 0 Hr 1 Hr 2 Hr 3 Hr 4 Hr 5 Hr 6Hr 7 Hr 8 Urine Output ATII 41.7(51.7) 28.6(32.4) 45.9(96.5) 31.1(58.0)33.7(67.1) 42.6(59.8) 35.9(50.0) 34.4(57.2) 36.1(38.3) 27.2(33.3)23.8(27.3) Placebo 29.5(69.8) 12.4(23.2) 23.5(41.8) 17.5(25.7)17.0(32.0) 16.3(24.6) 17.0(34.7) 16.8(30.4) 14.8(26.3) 18.0(27.3)23.0(34.4) Cardiac Output ATII 7.0(2.7) 6.0(3.1) 6.6(2.6) 6.3(2.5)6.2(2.5) 5.9(2.7) 6.5(2.4) 6.1(2.6) 6.7(3.3) 6.3(2.9) 7.5(3.1) Placebo6.3(1.2) 6.9(2.5) 6.5(1.7) 6.9(1.8) 6.4(1.7) 6.8(3.0) 7.3(2.2) 6.8(1.5)7.3(1.8) 6.9(2.5) 7.0(2.3) CVP ATII 12.7(5.5)  12.9(7.0)  14.1(8.9) 14.8(7.3)  14.6(7.4)  14.8(8.8)  11.7(3.9)  12.1(4.8)  10.0(2.9) 12.6(4.8)  11.8(3.8)  Placebo 16.0 (3.0)  9.7(2.1) 12.6(7.8)  15.7(9.3) 17.3(9.0)  15.7(7.3)  16.4(8.6)  16.3(6.7)  14.2(6.5)  14.4(7.1) 13.2(5.6)  MAP ATII 71.2(13.6) 72.3(11.2) 68.8(7.0)  74.8(8.4) 69.8(8.6) 73.1(12.5) 75.3(14.2) 68.9(8.1)  73.0(10.5) 72.3(11.9)73.6(11.5) Placebo 71.2(9.2)  71.8(6.5)  73.0(12.6) 72.8(9.5) 67.8(6.6)  70.1(6.4)  71.3(7.8)  73.0(4.7)  75.9(9.4)  74.0(10.6)74.5(13.2) Lactate ATII 4.6(3.1) 5.2(4.1) Placebo 7.1(5.2) 5.7(3.9)Legend: Urine Output (cc). Cardiac Output (liters/min), CVP = centralvenous pressure; all variables-mean (s.d.), * denotes p < 0.05.

Further studies showed that subjects who are administered as little as1.25 or 2.5 ng/kg/min of angiotensin II show an increase in bloodpressure and maintain it, even in the absence of, or with very low dosesof, a catecholamine, such as norepinephrine. The standard of care forpatients with high-output shock is to maintain the mean arterialpressure at 65 mm of Hg with catecholamines and/or vasopressin. Theinability to maintain blood pressure in mammals for an extended periodof time is uniformly fatal. In the study illustrated in FIG. 4, thestandard of care was to administer norepinephrine. 20% of the patientsresponded to very low doses of angiotensin II (1.25-2.5 ng/kg/min) suchthat the MAP rose markedly. Per standard protocol, the catecholaminedose was decreased as pushing the MAP above normal in high-output shockpatients is non-standard. Even as the catecholamines were completelyweaned off, the low dose of Angiotensin II resulted in a MAP>85 mm ofHg.

ATII was shown to be an effective pressor agent at a dose range of 1-40ng/kg/min. More specifically, a starting dose of 2-10 ng/kg/min may bean appropriate starting dose in the treatment of high output shock whenused in conjunction with standard-of-care vasopressors.

While all patients in the study had a response to the ATII infusion,significant heterogeneity was observed. Of the ten patients who receivedATII, two had a modest response, while two were exquisitely sensitive toATII, which was an unexpected finding. In the two highly sensitivepatients, the norepinephrine infusion was titrated off per protocol, andthe ATII dose was at its lowest allowable dose of 5 ng/kg/min and thepatients remained hypertensive with MAP of >90 mm Hg despitenorepinephrine titrated off. Since hypertension is not part of ourstandard of care, the investigators halted the infusion, and the ATIIwas weaned off. In both cases the need for norepinephrine was rapidlyreestablished.

ATII appears to have synergy with other vasopressors (i.e.catecholamines and vasopressin). It appears that for patients whorequire norepinephrine and are tachycardic, ATII is particularly useful.For patients with severe hypotension, lower doses of multiplevasopressors with differing mechanisms of action may be more efficaciousand less toxic than high doses of one type of vasopressors (i.e.,catecholamines).

Among the multiple strengths of the present study are the following.First, the study was a randomized, double-blind controlled trial with anappropriate placebo control arm. Secondly, it was of pragmatic design,as it was the intent of the investigators to enroll patients receivingstandard-of-care treatment for high output shock. As such, all patientshad received a priori appropriate monitoring and therapeuticinterventions (including central venous lines, bladder catheters,arterial lines, and cardiac output monitoring devices). There was noadditional need for any specialized equipment of procedures prior toenrollment in the study. Thirdly, all enrolled patients had a documentedneed for high dose vasopressor therapy despite volume therapy, asevidenced by the cardiac index entry criteria. This is in keeping withthe current practice of addressing volume responsiveness in ahypotensive patient prior to initiation of vasopressor therapy. Finally,as part of the study protocol, a data safety monitor was employed, whohad the ability to unblind data and evaluate for adverse events as wellas halt the study, neither of which occurred.

The initiation of an ATII infusion in patients receiving norepinephrinefor septic shock resulted in a marked decrease in norepinephrine doses.ATII improved blood pressure in patients with high-output shock andmultiple vasopressors. ATII is an effective as a pressor agent in thetreatment of high output shock. Initial dosing can range from between2-10 ng/kg/min. Finally, ATII was shown to be well-tolerated.

Another aspect of the present invention relates, inter alia, toformulations (compositions, pharmaceutical compositions) comprisingangiotensin II in combination with one or more further peptides and/orcompounds, as well as methods for the use of those formulations in thetreatment of subjects in need of increased blood pressure (havinghypotension), e.g. as present in a variety of types of shock, such as,e.g., high output shock, septic shock or shock from other forms of shocksuch as cardiac arrest or cardiogenic shock. The formulations can alsobe used to treat other conditions, including i.a. acute kidney injury,hepato-renal syndrome (HRS) or variceal bleeding.

Particular embodiments include, e.g., angiotensin II in combination withother vasopressor peptides or analogues or derivatives thereof; andangiotensin II in combination with other vasopressor peptides or analogsor derivatives thereof and a catecholamine. Additional embodimentsinclude any of the preceding peptides or combinations further incombination with the compound methylene blue, also referred to herein as3,7-bis(dimethylamino)-phenothiazin-5-ium chloride or MB.

The present inventor has found, unexpectedly, that the use ofangiotensin II in combination with other vasopressors, such asvasopressin and/or vasopressin analogues and/or with catecholaminesallows for the use of much lower doses of the vasopressin and/orvasopressin analogues and/or catecholamine than are currentlyadministered as the standard of care, thereby reducing side effects ofthose agents and increasing efficiency. Methylene blue, also, exhibits asynergistic effect with any of these agents. That is, a combination oflow doses of two or more of these agents (in combination withangiotensin II) is more effective than conventional large doses of oneof the agents.

One aspect of the invention is a composition comprising angiotensin IIor an analogue thereof and at least one additional vasopressor and/ormethylene blue. Such combinations are sometimes referred to herein as“multicomponent compositions of the invention.”

In embodiments of the invention, the multicomponent compositioncomprises the following combinations of agents:

angiotensin II+vasopressin

angiotensin II+vasopressin+norepinephrine

angiotensin II+vasopressin+any catecholamine,

methylene Blue (MB)+angiotensin II

MB+angiotensin II+vasopressin

MB+angiotensin II+vasopressin+norepinephrine

MB+angiotensin II+vasopressin+any catecholamine

angiotensin II+terlipressin

angiotensin II+terlipressin+norepinephrine

angiotensin II+terlipressin+any catecholamine

methylene blue (MB)+angiotensin II

MB+angiotensin II+terlipresssin

MB+angiotensin II+terlipresssin+norepinephrine

MB+angiotensin II+terlipressin+any catecholamine

Another aspect of the invention is a pharmaceutical compositioncomprising a multicomponent composition as above and a pharmaceuticallyacceptable carrier.

Another aspect of the invention is a kit comprising, in one or morecontainers, angiotensin II and at least one additional vasopressorand/or methylene blue.

Another aspect of the invention is a method (e.g. a method for treatinga subject in need thereof, e.g. in need of increased blood pressure,such as shock), comprising administering to the subject atherapeutically effective amount of a multicomponent compositioncomprising angiotensin II and at least one additional vasopressor and/ormethylene blue, or with a pharmaceutical composition comprising thecomposition and a pharmaceutically acceptable carrier.

As was discussed above, agents such as the agents noted above can beadministered conjointly, which refers to any form of administration oftwo agents (or more) such that the second agent (or additional agent(s))is administered while the previously administered agent is stilleffective in the body (e.g., the two (or more) agents are simultaneouslyeffective in the patient). For example, two (or more) agents can beadministered either in the same formulation or in separate formulations,either concomitantly or sequentially. In certain embodiments, thedifferent agents can be administered within one hour, 12 hours, 24hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, anindividual who receives such treatment can benefit from a combinedeffect of the two or more agents. Two, three or four of the agentsindicated above can be administered conjointly.

A composition for administering combinations of two or more of theindicated agents can take any of a variety of forms. For example, two ofmore of the agents can be packaged together and administered together.In another embodiment, one or more of the agents in a composition ispackaged separately from the others, so it can be administeredindependently from the others, e.g., in a separate I.V. line. In thismanner, for example, doses of the individual agents can be controlledindividually. In one embodiment, for example in an emergency when apatient has had a cardiac arrest, the subject can be administered acombination of, e.g., angiotensin II, vasopressin and a catecholamineoutside of the hospital, such as in an ambulance. Later, after thepatient has arrived at the hospital, more refined doses and combinationsof agents can be administered.

Angiotensin II is discussed above. The dose of angiotensin II which isadministered to a subject in the context of multicomponentadministration can be determined (titrated when used in combination witha catecholamine, vasopressin and/or methylene blue) by a method asdiscussed elsewhere herein. Generally, the dose of angiotensin IIadministered to a subject in the context of multicomponentadministration is from about 1 or 1.25 ng/kg to about 20 ng/kg, fromabout 1 or 1.25 ng/kg/min to about 10 ng/kg/min, from about 1 or 1.25ng/kg/min to about 5 ng/kg/min, from about 0.25 ng/kg/min to about 20ng/kg/min, from about 0.25 ng/kg/min to about 10 ng/kg/min, or fromabout 0.25 ng/kg/min to about 5 ng/kg/min. In embodiments of theinvention, the dose is about 0.25 ng/kg/min, about 0.5 ng/kg/min, about1 ng/kg/min, about 1.25 ng/kg/min, about 1.5 ng/kg/min, about 2ng/kg/min, about 2.5 ng/kg/min, about 3 ng/kg/min, about 3.5 ng/kg/min,about 4 ng/kg/min, about 4.5 ng/kg/min, about 5 ng/kg/min, about 5.5ng/kg/min, about 6 ng/kg/min, about 7.5 ng/kg/min or about 10 ng/kg/min.

As was discussed above, the catecholamines that can be used in a methodof the invention include, i.a., norepinephrine, epinephrine, dopamine orphenylephrine or ephedrine. The dose of a catecholamine which isadministered to a subject in the context of multicomponentadministration can be determined (titrated when used in combination withangiotensin II, vasopressin and/or methylene blue) by a method asdiscussed elsewhere herein. Generally, the dose of a catecholamine whichis administered to a subject in the context of multicomponentadministration is equivalent to a dose of norepinephrine of from about0.01 mg/kg/min to about 0.1 mcg/kg/min (e.g. about 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 mcg/kg/min). Equivalent dosesof catecholamines are summarized above.

Vasopressin or any of a variety of suitable analogues or derivativesthereof can be used in a method of the present invention; suitableanalogues or derivatives will be evident to a skilled worker. Amongthese suitable analogues or derivatives are: terlipressin, argipressin,desmopressin, felypressin, lypressin, and ornipressin. One suitableanalogue, terlipression, is a synthetic triglycyl-lysine derivative ofvasopressin, which is an inactive prodrug. It has pressor andantidiuretic effects. Following intravenous (IV) injection, lysinevasopressin is released following the enzymatic cleavage of 3 glycylmoieties. The dose of vasopressin or an analogue thereof which isadministered to a subject in the context of multicomponentadministration can be determined (titrated when used in combination withangiotensin II, a catecholamine and/or methylene blue) by a method asdiscussed elsewhere herein. Generally, the dose of vasopressin which isadministered to a subject in the context of multicomponentadministration is about 0.01 U/min to about 0.04 U/min (e.g. about 0.01,about 0.02, about 0.03 or about 0.04 U/min). Generally, the dose ofterlipressin which is administered to a subject in the context ofmulticomponent administration is about 0.1 mg to about 1 mg, e.g. about0.1, 0.3, 0.7 or 1 mg, for about 4-6 hours.

Methylene blue (MB) can also be administered to a subject in amulticomponent administration method of the invention. Methylene blue isa selective inhibitor of guanylate cyclase, a second messenger involvedin nitric oxide-mediated vasodilation, and as such can enhance theaction of vasopressors. As an experimental pharmaceutical drug, theInternational Nonproprietary Name (INN) of methylene blue ismethylthioninium chloride. The dose of methylene blue which isadministered to a subject in the context of multicomponentadministration can be determined (titrated when used in combination withangiotensin II, a catecholamine, and/or methylene blue) by a method asdiscussed elsewhere herein. Generally, the dose of methylene blue whichis administered to a subject in the context of multicomponentadministration is about 0.01 to about 3 mg/kg every 2-8 hours.

Treatment with the low doses of two or more of these agents (includingangiotensin II) as noted above is more effective than conventional largedoses of one of the agents. For example, the standard of care foradministering a catecholamine by itself is in the range of about 0.01 toabout 0.3 mcg/kg/min, and the standard of care for administeringvasopressin in the range of about 0.01 to about 0.08 U/min. By combiningtwo or more of these components, a reduction of at least about 20% toabout 25% of any of these over the conventional standard of care,represents a significant advantage.

The present inventor shows herein that smaller doses of multiplevasoconstrictors work more efficiently than large doses of onevasopressor. Without wishing to be bound by any particular mechanism, itis suggested that this synergy is due, at least in part, to theobservation that vasopressin and angiotensin II affect differentvascular beds differentially than catecholamines, so by attackingdifferent targets, the agents act synergistically. For example,vasopressin tends to cause more mesenteric vasoconstriction thancatecholamines, so if a patient develops mesenteric ischemia,vasopressin/vasopressin analogues are usually stopped. In diseases likehepato-renal syndrome, the disease problem is mesenteric vasodilation,and vasopressin/vasopressin analogues are deployed to counteract thatspecific regional vasodilation.

One embodiment of the invention is a kit which comprises angiotensin IIand one of more of the agents vasopressin or an analogue thereof (e.g.terlipressin), and/or a catecholamine, and/or methylene blue. The agentsin the kit can be present in individual containers (e.g. vials), or twoor more agents can be present together in a single container. In oneembodiment, each container contains a unit dose of the agent. In otherembodiments, multiple dose units are present in each container. Theagents may be in liquid form or they may be in solid (e.g. powdered orlyophilized form), which can be reconstituted with saline or acomparable diluent solution before administration to a patient. Forexample, in one embodiment, the following agents are present in solidform in a single vial: about 0.25 mg to about 1 mg of the catecholamineepinephrine; about 10 U to about 40 U of vasopressin; and about 0.01micrograms to about 100 micrograms of angiotensin II. These componentsare then suspended in a suitable volume of a diluent before use.Suitable diluents and suitable amounts of other combinations of agentswill be evident to a skilled worker.

Kits of the invention may comprise instructions for performing a method,such as methods for reconstituting solid forms of agents or for dilutingliquid forms. Other optional elements of a kit of the invention includesuitable buffers or other diluents for reconstituting solid forms of, orfor diluting liquid forms of, the agents; or packaging materials. Thereagents of the kit may be in containers in which the reagents arestable, e.g., in lyophilized or powdered form or stabilized liquids.

A composition or pharmaceutical composition of the invention can be usedto treat any of a variety of diseases or conditions. These include,e.g., shock, including septic shock, shock from cardiac arrest orcardiogenic shock, or high output shock. Other indications include acutekidney injury, hepato-renal syndrome (HRS) and variceal bleeding.

Acute Kidney Injury

Patients with inflammation/sepsis can develop acute kidney injury (AKI)due in part to vasodilation of glomerular efferent arteriole whichcauses intra-glomerular hypotension and loss of GFR. The parenteral useof agents such as angiotensin II and vasopressin causes efferentarteriole vasoconstriction, thereby mitigating this effect.

Hepato-Renal Syndrome (HRS)

HRS is a disease wherein mesenteric vasodilation is so profound thatblood flows preferentially to the gut and away from the kidney. HRSoccurs typically in patients with liver cirrhosis. Angiotensin II aloneor in combination with vasopressin and/or a catecholamine causesvasoconstriction and consequent improvement in renal function. Inaddition, patients with cirrhosis often have vasodilation due toincreased nitric oxide levels, and thus can be treated with methyeleneblue in conjunction with angiotensin II and a vsaopressin (e.g.terlipressin) and/or a catecholamine.

Variceal Bleeding

Patients with portal hypertension, usually from cirrhosis, tend to bleedfrom their esophageal varices. It is desirable to deploy drugs whichdecrease portal pressures. Treatment with a composition of the inventionresults in reduced bleeding, so fewer transfusions or procedures to stopbleeding are required, and death from the condition is reduced.

REFERENCES

-   1. Vincent J L, De Backer D: Circulatory shock. N Engl J Med 2013,    369(18):1726-1734.-   2. Myburgh J A, Higgins A, Jovanovska A, Lipman J, Ramakrishnan N,    Santamaria J, CAT Study investigators: A comparison of epinephrine    and norepinephrine in critically ill patients. Intensive Care Med    2008, 34(12):2226-2234.-   3. Rona G: Catecholamine cardiotoxicity. J Mol Cell Cardiol 1985,    17(4):291-306.-   4. Morelli A, Ertmer C, Westphal M, Rehberg S, Kampmeier T, Ligges    S, Orecchioni A, D'Egidio A, D'Ippoliti F, Raffone C, Venditti M,    Guarracino F, Girardis M, Tritapepe L, Pietropaoli P, Mebazaa A,    Singer M: Effect of heart rate control with esmolol on hemodynamic    and clinical outcomes in patients with septic shock: a randomized    clinical trial. JAMA 2013, 310(16):1683-1691.-   5. Russell J A, Walley K R, Singer J, Gordon A C, Hebert P C, Cooper    D J, Holmes C L, Mehta S, Granton J T, Storms M M, Cook D J,    Presneill J J, Ayers D, VASST Investigators: Vasopressin versus    norepinephrine infusion in patients with septic shock. N Engl J Med    2008, 358(9):877-887.-   6. Basso N, Terragno N A: History about the discovery of the    renin-angiotensin system. Hypertension 2001, 38(6):1246-1249.-   7. Struthers A D, MacDonald T M: Review of aldosterone- and    angiotensin II-induced target organ damage and prevention.    Cardiovasc Res 2004, 61(4):663-670.-   8. Jackson T, Corke C, Agar J: Enalapril overdose treated with    angiotensin infusion. Lancet 1993, 341(8846):703.-   9. Trilli L E, Johnson K A: Lisinopril overdose and management with    intravenous angiotensin II. Ann Pharmacother 1994, 28(10):1165-1168.-   10. Vincent J L, Moreno R, Takala J, Willatts S, De Mendonca A,    Bruining H, Reinhart C K, Suter P M, Thijs L G: The SOFA    (Sepsis-related Organ Failure Assessment) score to describe organ    dysfunction/failure. On behalf of the Working Group on    Sepsis-Related Problems of the European Society of Intensive Care    Medicine. Intensive Care Med 1996, 22(7):707-710.-   11. Newby D E, Lee M R, Gray A J, Boon N A: Enalapril overdose and    the corrective effect of intravenous angiotensin II. Br J Clin    Pharmacol 1995, 40(1):103-104.-   12. Wray G M, Coakley J H: Severe septic shock unresponsive to    noradrenaline. Lancet 1995, 346(8990):1604.-   13. Whiteley S M, Dade J P: Treatment of hypotension in septic    shock. Lancet 1996, 347(9001):622.-   14. Ryding J, Heslet L, Hartvig T, Jonsson V: Reversal of    ‘refractory septic shock’ by infusion of amrinone and angiotensin II    in an anthracycline-treated patient. Chest 1995, 107(1):201-203.-   15. Thomas V L, Nielsen M S: Administration of angiotensin II in    refractory septic shock. Crit Care Med 1991, 19(8):1084-1086.-   16. Yunge M, Petros A: Angiotensin for septic shock unresponsive to    noradrenaline. Arch Dis Child 2000, 82(5):388-389.-   17. Correa T D, Jeger V, Pereira A J, Takala J, Djafarzadeh S, Jakob    S M: Angiotensin II in Septic Shock: Effects on Tissue Perfusion,    Organ Function, and Mitochondrial Respiration in a Porcine Model of    Fecal Peritonitis. Crit Care Med 2014. August; 42(8): e550-9-   18. Wan L, Langenberg C, Bellomo R, May C N: Angiotensin II in    experimental hyperdynamic sepsis. Crit Care 2009, 13(6):R190.-   19. Goldsmith S R, Hasking G J: Effect of a pressor infusion of    angiotensin II on sympathetic activity and heart rate in normal    humans. Circ Res 1991, 68(1):263-268.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodifications of the invention to adapt it to various usage andconditions and to utilize the present invention to its fullest extent.The preceding specific embodiments are to be construed as merelyillustrative, and not limiting of the scope of the invention in any waywhatsoever. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, numerousequivalents to the compounds and methods of use thereof describedherein. Such equivalents are considered to be within the scope of thisinvention and are covered by the following claims. Those skilled in theart will also recognize that all combinations of embodiments describedherein are within the scope of the invention.

The entire disclosure of all applications, patents, and publicationscited above, including US provisional applications U.S. ProvisionalApplication No. 61/917,576, filed Dec. 18, 2013, and U.S. ProvisionalApplication No. 61/955,706, filed Mar. 19, 2014, including the figures,are hereby incorporated in their entirety by reference, particularlywith regard to the disclosure for which they are referenced.

What is claimed is:
 1. A method of treating a human patient sufferingfrom low blood pressure, comprising administering angiotensin II to thepatient at an initial rate of about 20 ng/kg/min and titrating the ratedown, wherein the angiotensin II is administered at a rate sufficient toachieve and/or maintain a mean arterial pressure (MAP) of about 65 mm Hgor above.
 2. The method of claim 1, wherein the rate is titrated down toabout 15 ng/kg/min.
 3. The method of claim 1, wherein the rate istitrated down to about 10 ng/kg/min.
 4. The method of claim 1, whereinthe rate is titrated down to about 5 ng/kg/min.
 5. The method of claim1, wherein the rate is titrated down to about 1.25 ng/kg/min.
 6. Themethod of claim 1, wherein the patient has shock, distributive shock,cardiogenic shock, sepsis, or acute kidney injury.
 7. The method ofclaim 6, wherein the angiotensin II consists of the amino acid sequenceset forth in SEQ ID NO:
 1. 8. The method of claim 7, wherein theangiotensin II is administered intravenously.
 9. The method of claim 6,wherein the rate is titrated down to about 15 ng/kg/min.
 10. The methodof claim 6, wherein the rate is titrated down to about 10 ng/kg/min. 11.The method of claim 6, wherein the rate is titrated down to about 5ng/kg/min.
 12. The method of claim 6, wherein the rate is titrated downto about 1.25 ng/kg/min.
 13. The method of claim 1, wherein theangiotensin II consists of the sequence of SEQ ID NO:
 1. 14. The methodof claim 6, wherein the rate is titrated down to about 0.25 ng/kg/min.15. The method of claim 1, wherein the rate is titrated down to about0.25 ng/kg/min.
 16. A method of achieving target mean arterial pressure(MAP) of at least about 65 mmHg in a human patient experiencing lowblood pressure, comprising administering angiotensin II to the patientat an initial rate of about 20 ng/kg/min and titrating the rate down.17. The method of claim 16, wherein the rate is titrated down to about15 ng/kg/min.
 18. The method of claim 16, wherein the rate is titrateddown to about 10 ng/kg/min.
 19. The method of claim 13, wherein the rateis titrated down to about 5 ng/kg/min.
 20. The method of claim 13,wherein the rate is titrated down to about 1.25 ng/kg/min.
 21. Themethod of claim 13, wherein the patient has shock, distributive shock,cardiogenic shock, sepsis, or acute kidney injury.
 22. The method ofclaim 21, wherein the angiotensin II consists of the amino acid sequenceset forth in SEQ ID NO:
 1. 23. The method of claim 22, wherein theangiotensin II is administered intravenously.
 24. The method of claim21, wherein the rate is titrated down to about 15 ng/kg/min.
 25. Themethod of claim 21, wherein the rate is titrated down to about 10ng/kg/min.
 26. The method of claim 21, wherein the rate is titrated downto about 5 ng/kg/min.
 27. The method of claim 21, wherein the rate istitrated down to about 1.25 ng/kg/min.
 28. The method of claim 21,wherein the rate is titrated down to about 0.25 ng/kg/min.
 29. Themethod of claim 13, wherein the angiotensin II consists of the sequenceof SEQ ID NO:
 1. 30. The method of claim 16, wherein the rate istitrated down to about 0.25 ng/kg/min.